Protein based biopharmaceuticals show great promise in providing more specific and tissue specific, or cell specific drug treatments against serious diseases (for overview see “Recombinant Protein Drugs” Ed. P. Buckel 2001).
Numerous examples in the prior art and applications have demonstrated the use of microorganisms such as bacteria, and animal cells for the production of such biopharmaceuticals, of which insulin is a notable example.
Many examples in the literature have demonstrated the utilization of transgenic plants or plant cell cultures for expression and manufacturing of high-value heterologous polypeptides or biopharmaceuticals. Such plant-based manufacturing process may be called molecular farming.
Production of valuable proteins can be made more economical by the use of plants as production organisms. The cultivation cost for plants used as host organisms for protein manufacturing can be considerably lower compared to most production systems based on bioreactors, such as prokaryotic production systems, animal cell cultivation and so forth. However, for all of the above production systems, purification of heterologous proteins remains a demanding and costly task. Thus, for plant-based production systems, downstream processing generates most of the production costs in the manufacturing of high-value heterologous proteins.
Protein purification and isolation is a key process in downstream processing of proteins accumulated and produced in a variety of host organisms by the use of gene technology. The purification of proteins from the host organisms can be quite laborious, complex and expensive. A variety of chromatographic strategies are used commercially for separation and purification of proteins of interest from production host organisms. The chromatographic strategies may rely on physicochemical differences between contaminating or endogenous proteins and the heterologous protein of interest, such as in size, solubility, charge, hydrophobicity, and affinity.
Combinations of chromatographic strategies consisting of multiple steps, require several expensive chromatography matrices and the necessary hardware consisting of columns, control units and so forth, and are accompanied by product yield-losses at each step, and consequently, economical losses. In addition to the chromatographic steps involved, downstream processing typically involves multiple filtering and centrifugation steps. As a result, the cost of purification and downstream processing may become prohibitive for the purification of a protein based biotechnological product. As for a number of protein-based products of lesser value, such as industrial proteins, the cost of downstream processing can be inhibitory for their use and marketing, resulting in crude and poorly defined products. For most biotechnology products purification costs are certainly a major proportion of the manufacturing costs.
The cost of specialized chromatography matrices effecting the separation of a protein of interest from contaminants is high as a result of complex coupling chemistry involved in their production. Some common chromatography methods are ion exchange chromatography, hydrophobic interaction chromatography and reversed phase chromatography. Ligands coupled to a matrix may include a variety of diethylaminoethyl- or quaternary aminoethyl groups as anion exchangers and carboxymethyl substituents and sulfonate groups and phosphates as cation exchangers of varying strength (Scopes, 1993), hydrophobic alkyl, phenyl or butyl groups coupled to crosslinked agarose matrices for hydrophobic interaction chromatography (Hydrophobic Interaction Chromatography (HIC) nr. 18-1020-90, Amersham Pharmacia Biotech), or hydrophobic n-alkyl groups as ligands grafted to a porous, insoluble beaded matrices composed of silica or synthetic organic polymers (Reversed phase Chromatography (RPC), Code nr. 18-11134-16, Amersham Pharmacia Biotech) The chemical complexity of these matrices may cause unwanted leaching of ligands and other substances from the matrix during a purification procedure, where necessary preventive measures, monitoring, or the removal of leachates from the protein of interest add to the already high cost of downstream processing.
Affinity chromatography is among the most powerful purification principles as it is based on specific affinity between an agent and a specific ligand, often mimicking a natural protein-ligand interaction. Several different kinds of affinity adsorbents are available, some highly specific for a particular protein, others binding to classes of proteins rather than particular proteins. In many cases affinity chromatography implies the use of an immobilized ligand to an adsorbent that specifically selects out proteins binding to that ligand. The coupling of ligands to affinity adsorbents involves the use of coupling chemistry such as cyanogen bromide-, tosyl-, or vinylsulfone-activation of adsorbents. The ligand coupled to the column matrix may or may not be of proteinaceous origin. Examples of the former are, but not limited to, immobilized protein A or protein G having affinity for ?-globulin, therefore being useful in the purification of antibodies, and lectins with affinity for glycoproteins. As an example of the latter, immobilized glutathione coupled to matrix binds fusion proteins containing a glutathione S-transferase domain. Immobilized metal affinity chromatography (IMAC) is based on immobilization of metal-chelating ligands to a matrix and relies on the formation of weak coordinate bonds between metal ions immobilized on a column and basic groups on proteins, mainly histidine residues. Commercial cloning vectors provide for cloning of a cDNA in frame with a string of histidine residues—a His-tag, that enables the purification of the resultant fusion protein with IMAC. Although widely used for small-scale purification of proteins, IMAC is a non-specific but selective method, as native histidine residues in contaminating proteins can lead to binding in IMAC (Scopes 1993). Several different kinds of tags or binding domains are available in commercial expression vectors resulting in fusion proteins where the tag/binding domain binds the fusion protein to a ligand coupled onto a column matrix.
High specificity of protein binding can be achieved with these matrix-ligand systems. In the cases mentioned above, complex coupling chemistry is involved to immobilize a ligand onto an inert matrix. Consequently, the cost of an affinity matrix can often become inhibitory to industrial scale applications of this powerful technique. Furthermore, as with most other types of chromatography methods, the stability of the coupling of the ligand to the matrix becomes an issue and leaching is of great concern. Heavy metal leaching in IMAC can cause unacceptable and serious contamination in many sensitive purification processes for bioactive proteins, and may inactivate proteins being purified (Scopes 1993).
It is of particular relevance for the present invention that it is not uncommon that the binding affinity of a protein to its ligand is so strong that conditions for elution, to disrupt the ligand-protein binding, require drastic conditions that partly denature the valuable protein being purified. A non-limiting example of this is the elution of antibodies from Protein A-affinity matrix, requiring denaturing at low pH to release the antibody from the column. Including a denaturing step in a protein purification process is undesirable due to the risk of loss of activity of the purified protein, the addition of an extra step for refolding the protein and subsequent activity analysis requirements for the refolded protein product, and the added cost involved.
To enable the use of affinity-based chromatography for large scale purification from plants, it is highly desirable to develop a non-denaturing protein purification process that is simpler and more economic than the current measures commercially available, with less coupling chemistry involved and compatible with the quality requirements of the pharmaceutical industry standards.
Plant-based production of proteins shows great promise for large scale manufacturing of proteins in an economic manner, as has been shown by examples in literature (for overview see Hammond 1999). The cultivation costs involved in molecular farming with plants are considerably lower than with traditional bioreactor-based methods. Whereas upstream events in plant-based production look particularly promising, the downstream processing is facing the same challenges as the rest of the protein production industry.
Polysaccharides and polysaccharide binding proteins may be used in conjunction for the design of an affinity chromatography step (see, e.g., Boraston et al., 2001).
In an example of prior art (U.S. Pat. No. 6,331,416) Shani et al. describe a method of expressing a recombinant protein with a polysaccharide binding domain that binds to the poorly defined cellulose in the host plant cell walls, and a protein purification process utilizing the affinity of this protein to host plant cellulose, resulting in a cell wall-protein complex that can be separated from soluble contaminating proteins. The strength of binding can be such that releasing the protein from the cellulosic host plant matter may require drastic conditions that denature the protein, having negative effects on the activity of the recombinant proteins being purified. Thus, although presenting a way to purify proteins from plant material the harsh conditions required for disrupting the ligand/cell-wall binding involve complications and concerns comparable to those mentioned above for antibody-Protein A elution.
To enable the use of affinity-based chromatography for large scale purification from plants, it is highly desirable to develop a non-denaturing protein purification process that is simpler and more economic than the current measures commercially available, with less coupling chemistry involved and compatible with the quality requirements of the pharmaceutical industry standards.
The carbohydrate binding domain CBM9-2 is from the Thermotoga maritima Xylanase 10A (Winterhalter et al 1995: Mol. Microbiol. 15 (3), 431-444). The CBM9-2 genomic DNA sequence is available as GenBank Accession No. Z46264 and it belongs to the Family IX of CBM-s and has number of attractive properties for high-resolution affinity purification, including non-denaturing eluting conditions using 1M glucose as a eluent, and high specific affinity for amorphous as well as crystalline celluloses (Boraston et al. 2001: Biochemistry 40, 6240-6247).
Recovery and purification of expressed recombinant proteins from transgenic plants is probably the most critical factor in establishing plants as a practical alternative system for protein production. It is essential that the number of processing steps be minimized and that each step be carried out at much higher efficiencies. (Moloney 2000)
This emphasizes a recognized need for a downstream process for purification of heterologous proteins from transgenic plant material that is efficient, simple, and economical. Furthermore, there is a need for such a downstream process consisting of gentle, non-denaturing conditions for the protein of interest, (in particular, specific affinity purification methods with gentle elution conditions) in order to secure bioactivity of the protein of interest, and improve yields.
A non-denaturing protein purification process free from the limitations detailed above could significantly lower the production cost involved in the production of biopharmaceuticals from plants, and would be enabling for the purification of heterologous proteins of value for which downstream processing has been prohibitively complex and costly.